Scrub typhus is a neglected disease that threatens the 1 billion inhabitants of the Asia-Pacific rim and causes 1 million new infections annually. Its mortality rate can be as high as 50%. The disease was a significant cause of morbidity in World War II and the Vietnam conflict and presently threatens U.S. soldiers serving in the Middle East. The causative agent is the chigger-transmitted obligate intracellular bacterium, Orientia tsutsugamushi, which colonizes microvascular endothelial cells, macrophages, and neutrophils. The proposed work will advance understanding of how Orientia facilitates its survival in its diverse host cell types, a subject that has long-remained a metaphoric black box. The ankyrin repeat is the most common protein-protein interaction motif in nature. Many intracellular bacterial and viral pathogens translocate ankyrin repeat-containing proteins (Anks) into eukaryotic host cells. The Anks interact with target proteins to modulate host cell functions. O. tsutsugamushi encodes 38 Anks, several of which also carry another eukaryotic-like motif, the F-box, in their C-termini. We refer to these as Ank/F-box proteins. Eukaryotic F-boxes bind SKP1, a part of the SCF1 ubiquitin ligase complex, which catalyzes the transfer of ubiquitin onto proteins to target them for degradation in the proteasome. The bipartite structure of Ank/F-box proteins conceivably enables them to bind protein substrates via their Ank domains and, using their F-box motifs, bind SKP1 to direct SCF1-mediated ubiquitination and subsequent proteasomal degradation of the substrates. We discovered that 5 of the 8 ank genes that Orientia expresses as it establishes infection in tissue culture cells encode Ank/F-box proteins. In Aim 1, we will evaluate our hypothesis that the Ank/F-box proteins interact with SKP1 to promote SCF1- mediated ubiquitination of host cell proteins. We will also identify the proteins that become ubiquitinated as a result of interacting with Ank/F-box proteins. Given its extensive Ank repertoire, we hypothesize that Orientia expresses specific Anks to establish infection in endothelial cells, macrophages, and neutrophils. In Aim 2, we will close an important knowledge gap by identifying the Anks that the pathogen expresses as it establishes infection in each of the 3 biologically relevant host cells.